Fluorescent substrate for detection of enzymatic activity of nitrile-related enzyme

ABSTRACT

The object of the present invention is to provide a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme. 
     The present invention provides a compound represented by formula (I) and a fluorescent substrate for detecting the enzymatic activity of a nitrile-related enzyme, which comprises the compound.

TECHNICAL FIELD

The present invention relates to a novel compound and a fluorescentsubstrate for detecting the enzymatic activity of a nitrile-relatedenzyme, which comprises the compound, as well as a method for detectingthe enzymatic activity of a test substance using the above compound.

BACKGROUND ART

In the production processes for compounds serving as source materials ofchemical products, enzymatic reactions are currently used due to theirhigh conversion and selectivity, particularly due to their highstereoselectivity in the case of optically active compounds. Forexample, enzymatic reactions catalyzed by nitrilase, nitrile hydratase,amidase and the like are used for production of nitrile compounds (e.g.,acrylamide, acrylic acid) serving as source materials of variouschemical products. Nitrilase is an enzyme converting a nitrile groupinto a carboxylic acid group through hydrolysis, nitrile hydratase is anenzyme converting a nitrile group into an amido group through hydration,and amidase is an enzyme converting an amido group into a carboxylicacid group through hydrolysis.

Microorganisms having these enzymes have been screened from the naturalenvironment including soil. Techniques commonly used for screeningpurposes involve enriching microorganisms which grow using nitrilecompounds or the like as a sole nitrogen or carbon source, and selectinga microorganism having enzymatic activity from among the resultingmicroorganisms. For verification of enzymatic activity, a nitrilecompound or an amide compound is reacted with each microorganism and theresulting product is analyzed with an instrument for high performanceliquid chromatography or gas chromatography, etc.

On the other hand, there is a research report showing thatmicroorganisms which can be isolated and cultured by currently usedtechniques constitute only less than 1% of the microorganisms present inthe natural environment (M. S. Rappe and S. J. Giovannoni, Annu. Rev.Microbiol., 57, 369 (2003)).

For this reason, another procedure (metagenome screening) has been usedin recent studies, which involves directly isolating genes(environmental DNAs or metagenomes) from the natural environment,instead of isolating microorganisms as in conventional techniques, andthen screening the isolated genes to select a useful enzyme gene. Theuse of this procedure requires techniques for preparing a very largenumber of metagenome-derived recombinants and efficiently selectingactive recombinants from among these recombinants. However, screeningwith an instrument for high performance liquid chromatography or gaschromatography is not high-throughput. Thus, there has been a demand fora novel technique which allows high-throughput screening.

Meanwhile, an invention of a fluorescent probe or fluorescent labelingagent is known as an invention directed to a fluorescent substance(WO2004/005917, WO2006/019105). However, the fluorescent substrate ofthe present invention useful for detection of enzymatic activity is notknown.

DISCLOSURE OF THE INVENTION

The present invention has been made under these circumstances, and theproblem to be solved by the present invention is to provide a novelcompound and a fluorescent substrate for detecting the enzymaticactivity of a nitrile-related enzyme, which comprises the compound, aswell as a method for detecting the enzymatic activity of a testsubstance using the above compound.

As a result of extensive and intensive efforts made to solve the aboveproblem, the inventors of the present invention have developed acompound whose fluorescence will vary by the action of a nitrile-relatedenzyme, e.g., a non-fluorescent (low fluorescent) substrate compoundwhich will emit fluorescence by the action of a nitrile-related enzyme.Moreover, the inventors of the present invention have also found thatthe enzymatic activity of a nitrile-related enzyme can be detected in asimple manner by measuring fluorescence from the substrate of thepresent invention. This finding led to the completion of the presentinvention.

Namely, the present invention is as follows.

-   (1) A method for detecting the enzymatic activity of a test    substance, which comprises the following steps:

(a) reacting a test substance with a compound whose fluorescence willvary by the action of a nitrile-related enzyme, and

(b) measuring the wavelength and intensity of fluorescence generated asa result of the reaction in step (a).

-   (2) The method according to (1) above, wherein the enzymatic    activity is the activity of one enzyme selected from the group    consisting of nitrilase, nitrile hydratase and amidase.-   (3) The method according to (1) above, wherein the compound whose    fluorescence will vary by the action of a nitrile-related enzyme is    a compound represented by the following formula (I), a salt thereof    or a hydrate thereof:

[wherein R¹ represents —CN, —CONH₂, —CH═CH—CN or —CH═CH—CONH₂, R²represents a C₁₋₄ alkyl group, R³ and R⁴ each independently represent ahydrogen atom, a halogen atom or a C₁₋₄ alkyl group, and R⁵ represents ahydrogen atom, a C₁₋₄ alkylcarbonyl group or a C₁₋₄alkylcarbonyloxymethyl group].

-   (4) The method according to (3) above, wherein R¹ is —CN or    —CH═CH—CN, and the nitrile-related enzyme is nitrilase or nitrile    hydratase.-   (5) The method according to (3) above, wherein R¹ is —CONH₂ or    —CH═CH—CONH₂, and the nitrile-related enzyme is amidase.-   (6) The method according to (1) above, wherein the fluorescence is    measured using flow cytometry.-   (7) A compound represented by the following formula (I), a salt    thereof or a hydrate thereof:

[wherein R¹ represents —CN, —CONH₂, —CH═CH—CN or —CH═CH—CONH₂, R²represents a C₁₋₄ alkyl group, R³ and R⁴ each independently represent ahydrogen atom, a halogen atom or a C₁₋₄ alkyl group, and R⁵ represents ahydrogen atom, a C₁₋₄ alkylcarbonyl group or a C₁₋₄alkylcarbonyloxymethyl group].

-   (8) A fluorescent substrate for detecting the enzymatic activity of    a nitrile-related enzyme, which comprises the compound according    to (7) above, a salt thereof or a hydrate thereof.-   (9) The substrate according to (8) above, wherein the    nitrile-related enzyme is at least one member selected from the    group consisting of nitrilase, nitrile hydratase and amidase.-   (10) The substrate according to (8) above, wherein R¹ is —CN or    —CH═CH—CN, and the nitrile-related enzyme is nitrilase or nitrile    hydratase.-   (11) The substrate according to (8) above, wherein R¹ is —CONH₂ or    —CH═CH—CONH₂, and the nitrile-related enzyme is amidase.-   (12) A kit for detecting the enzymatic activity of a nitrile-related    enzyme, which comprises the substrate according to any one of (8)    to (11) above.

By using the compound of the present invention, the enzymatic activityof a nitrile-related enzyme can be detected in a simple manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reaction path of a fluorescent substrate catalyzed bynitrile-related enzymes.

FIG. 2 shows the results of fluorescence measurement performed onfluorescent substrates.

FIG. 3 shows the detection results of a nitrile hydrataseactivity-induced increase in the fluorescence intensity of a fluorescentsubstrate.

FIG. 4 shows the detection results of a nitrilase activity-inducedincrease in the fluorescence intensity of a fluorescent substrate.

FIG. 5 shows the detection results of a nitrilase activity-inducedincrease in the fluorescence intensity of a fluorescent substrate.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below. Thefollowing embodiments are illustrated to describe the present invention,and it is not intended to limit the present invention only to theseembodiments. The present invention can be implemented in various modes,without departing from the spirit of the present invention. Moreover,this specification incorporates the contents disclosed in thespecification and drawings of Japanese Patent Application No.2010-119431 (filed on May 25, 2010), based on which the presentapplication claims priority.

1. Summary

The present invention relates to a compound whose fluorescence will varyby the action of a nitrile-related enzyme (hereinafter also referred toas “the compound of the present invention”) and a fluorescent substratefor detecting the enzymatic activity of a nitrile-related enzyme, whichcomprises the compound. The present invention also relates to a methodfor detecting the enzymatic activity of a test substance, whichcomprises reacting a test substance with the compound of the presentinvention and measuring the wavelength and intensity of fluorescencegenerated as a result of this reaction.

For use as a compound for detecting the enzymatic activity of anitrile-related enzyme, the inventors of the present invention haveattempted to synthesize a compound whose fluorescence will vary uponreaction with a nitrile-related enzyme. As a result of measuringfluorescence from the synthesized compound, the inventors of the presentinvention have found that fluorescence from a compound produced probablyas a result of the enzymatic reaction is increased when compared tofluorescence from the compound before being subjected to the enzymaticreaction. Based on this finding, the inventors of the present inventionhave considered that the synthesized compound is useful as a fluorescentsubstrate for detecting the enzymatic activity of a nitrile-relatedenzyme, and thus have completed the present invention.

2. Compound

In the context of the present invention, a “compound whose fluorescencewill vary by the action of a nitrile-related enzyme” is intended to meana compound whose fluorescence will vary when a nitrile group (—CN)contained in the compound is converted into an amido group (—CONH₂) or acarboxyl group (—COOH) by the action of the enzyme or when an amidogroup contained in the compound is converted into a carboxyl group bythe action of the enzyme. The compound of the present invention is notlimited in any way, as long as it has a fluorophore and a nitrile groupor an amido group, and as long as it is characterized in thatfluorescence from its fluorophore will vary when the nitrile group inthe compound is converted into an amido group or a carboxyl group orwhen the amido group in the compound is converted into a carboxyl group.

Moreover, although the mechanism of fluorescence variation by the actionof a nitrile-related enzyme differs from compound to compound, thefollowing explanation can be given for a compound represented by formula(I), by way of example.

This compound is substantially non-fluorescent or low fluorescent due toPeT (photo-induced electron transfer) before the reaction of anitrile-related enzyme. However, once its nitrile group or amido grouphas been hydrolyzed as a result of the enzymatic reaction, PeT will nolonger occur and fluorescence will be emitted. PeT is an electrontransfer reaction caused by photoexcitation.

For example, compound 1, which will be described later, is in asubstantially non-fluorescent state because fluorescence from itsexcited fluorophore (i.e., its xanthene ring moiety) is quenched due toPeT from the xanthene moiety to the benzene ring moiety. However, whenthe nitrile group in compound 1 is hydrolyzed by nitrilase to givecompound 5, PeT will no longer occur and compound 5 will emitfluorescence.

In the context of the present invention, a nitrile-related enzyme isintended to mean an enzyme that acts on nitrile-related compounds(compounds having a nitrile group and compounds having an amido group).Examples of such an enzyme include nitrilase, nitrile hydratase andamidase.

The “action” of such a nitrile-related enzyme is intended to meanhydration reaction or hydrolysis reaction.

A “variation” in fluorescence is intended to mean a shift in thefluorescence wavelength and/or a variation in the fluorescenceintensity. Determination of whether fluorescence has “varied” or not canbe accomplished based on the following criteria. If the fluorescenceintensity is increased, it is intended to mean a 1.5-fold or moreincrease, preferably a 2-fold or more increase, and more preferably a3-fold or more increase, while if the fluorescence intensity isdecreased, it is intended to mean a decrease to ⅔ or less, preferably adecrease to ½ or less, and more preferably a decrease to ⅓ or less.Likewise, if the fluorescence wavelength varies, it is intended to meana 10 nm shift, preferably a 30 nm shift, and more preferably a 60 nm ormore shift toward the longer or shorter wavelength side.

In the context of the present invention, examples of a compound whosefluorescence will vary by the action of a nitrile-related enzyme includea compound represented by the following formula (I).

In formula (I), the substituent represented by R¹ is not limited in anyway as long as it is a substituent capable of reacting with anitrile-related enzyme, as exemplified by —CN, —CONH₂, —CH═CH—CN or—CH═CH—CONH₂.

The substituent represented by R² may be exemplified by a C₁₋₄ alkylgroup.

The substituents represented by R³ and R⁴ may be exemplified by ahydrogen atom, a halogen atom or a C₁₋₄ alkyl group.

The substituent represented by R⁵ may be exemplified by a hydrogen atom,a C₁₋₄ alkylcarbonyl group or a C₁₋₄ alkylcarbonyloxymethyl group.

In the context of the present invention, a “C₁₋₄ alkyl group” isintended to mean a linear or branched alkyl group containing 1 to 4carbon atoms, and examples include a methyl group, an ethyl group, an-propyl group, an isopropyl group, a n-butyl group, an isobutyl group,a sec-butyl group, a tert-butyl group and so on, with a methyl groupbeing preferred.

In the context of the present invention, examples of a “C₁₋₄alkylcarbonyl group” include an acetyl group, an ethylcarbonyl group, apropionylcarbonyl group, an isobutylcarbonyl group and so on, with anacetyl group being preferred.

In the context of the present invention, examples of a “C₁₋₄alkylcarbonyloxymethyl group” include an acetoxymethyl group, anethoxymethyl group, a propionyloxymethyl group, an isobutyryloxymethylgroup and so on, with an acetoxymethyl group being preferred.

In the context of the present invention, examples of a “halogen atom”include a fluorine atom, a chlorine atom, a bromine atom and an iodineatom, with a fluorine atom or a chlorine atom being preferred.

The reason why R² is preferably a C₁₋₄ alkyl group, but not a hydrogenatom would be because if R² is a hydrogen atom, the electron density ofthe benzene ring is not sufficiently low to cause fluorophore quenchingby photo-induced electron transfer. Namely, if R² is a hydrogen atom,the fluorescence intensity is high even before enzymatic reaction andhence it cannot be expected to obtain a large variation in thefluorescence intensity upon enzymatic reaction catalyzed by anitrile-related enzyme. In contrast, if R² is a C₁₋₄ alkyl group, theelectron density of the benzene ring is sufficiently low to causefluorophore quenching by photo-induced electron transfer, so that it canbe expected to obtain a variation in the fluorescence intensity uponenzymatic reaction.

Typical examples are shown below for the compound represented by formula(I) (wherein Me represents a methyl group).

The above compound represented by formula (I) may form a salt or hydratethereof Any salt is possible as long as it has the effect of the presentinvention, and such a salt may be formed with either an acid or a base.

Examples of a salt with an acid include salts with inorganic acids(e.g., hydrochloride salt, hydrobromide salt, sulfate salt, phosphatesalt), as well as salts with organic acids such as formic acid, aceticacid, lactic acid, succinic acid, fumaric acid, maleic acid, citricacid, tartaric acid, benzoic acid, methanesulfonic acid, benzenesulfonicacid, p-toluenesulfonic acid, trifluoroacetic acid and so on.

Examples of a salt with a base include alkali metal salts (e.g., sodiumsalt, potassium salt), alkaline earth metal salts (e.g., calcium salt,magnesium salt), salts with organic bases such as trimethylamine,triethylamine, pyridine, picoline, dicyclohexylamine,N,N′-dibenzylethylenediamine and so on (organic amine salts), as well asammonium salts.

Alternatively, the compound of the present invention may not form anysalt, i.e., may be in a so-called free form.

The compound represented by formula (I) may be prepared in the followingmanner.

First, compound (a) to be used as a starting material can be obtained bybeing purchased from Tokyo Chemical Industry (TCI) Co., Ltd., Japan.Compound (a) is then reacted in DMF with Ac₂O and Cs₂CO₃ at roomtemperature for 1 hour to obtain compound (b). Compound (b) is thendissolved in 12 N HCl, and NaNO₂ is added dropwise thereto on an icebath, followed by stirring. After stirring for 30 minutes at 0° C., KIdissolved in water is added dropwise and the mixture is stirred for 10minutes. The mixture is returned to room temperature and reacted for 1hour to obtain compound (c). Compound (c) is further reacted in THF withKCN under reflux at about 80° C. for 3 hours in the presence of CuI andPd(PPh₃)₄ as a catalyst to obtain compound (d). Compound (d) is reactedin MeOH with MeONa at room temperature for 10 minutes to obtain compound(e). Finally, compound (e) is reacted in CsF and DMF with 1 equivalentof CH₃I at room temperature for about 1 hour to obtain compound 1.

3. Fluorescent Substrate

The above compound represented by formula (I), a salt thereof or ahydrate thereof may be used as a fluorescent substrate for detecting theenzymatic activity of a nitrile-related enzyme.

An explanation will be given below of the mechanism by which the abovecompound represented by formula (I) generates fluorescence.

The above compound represented by formula (I) is substantiallynon-fluorescent due to PeT (photo-induced electron transfer) before thereaction of a nitrile-related enzyme. However, once its nitrile group oramido group has been hydrolyzed as a result of the enzymatic reaction,PeT will no longer occur and fluorescence will be emitted. PeT is anelectron transfer reaction caused by photoexcitation.

For example, compound 1 is in a substantially non-fluorescent statebecause fluorescence from its excited fluorophore (i.e., its xanthenering moiety) is quenched due to PeT from the xanthene moiety to thebenzene ring moiety. However, when the nitrile group in compound 1 ishydrolyzed by nitrilase to give compound 5, PeT will no longer occur andcompound 5 will emit fluorescence (FIG. 1).

If fluorescence can be detected, such a result indicates the presence ofa nitrile-related enzyme in a test sample.

In the context of the present invention, examples of a nitrile-relatedenzyme include, but are not limited to, nitrilase, nitrile hydratase,and amidase.

A preferred nitrile-related enzyme is nitrilase or nitrile hydratase forcompounds represented by formula (I) in which R¹ is —CN or —CH═CH—CN,while a preferred nitrile-related enzyme is amidase for compoundsrepresented by formula (I) in which R¹ is —CONH₂ or —CH═CH—CONH₂.

4. Detection Method for Enzymatic Activity

The present invention provides a detection method for enzymaticactivity. More specifically, the present invention provides a method fordetecting the enzymatic activity of a test substance, which comprisesthe steps of: (a) reacting a test substance with a compound whosefluorescence will vary by the action of a nitrile-related enzyme; and(b) measuring the wavelength and intensity of fluorescence generated asa result of the reaction in step (a).

In the context of the present invention, the test substance is notlimited in any way, as long as it is a protein having the enzymaticactivity of a nitrile-related enzyme or is predicted to contain DNAencoding such a protein. Examples include substances contained inmetagenomic libraries obtained from the natural environment or mutatedenzyme gene libraries, etc. Further, such test substances encompass notonly DNAs and proteins, but also cells producing these proteins.Examples of such cells include bacteria, fungi (e.g., yeast, filamentousfungi), plant cells, animal cells and so on. These cells furtherencompass cells which have been transformed to express a protein havingthe enzymatic activity of a nitrile-related enzyme. Examples of thesetransformed cells include, but are not limited to, microbial cells forwhich host vector systems have been developed, as exemplified bybacteria of the genera Escherichia, Bacillus, Pseudomonas, Serratia,Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus,Rhodococcus and Streptomyces, yeast of the genera Saccharomyces,Kluyveromyces, Schizosaccharomyces, Zygosaccharomyces, Yarrowia,Trichosporon, Rhodosporidium, Pichia and Candida, filamentous fungi ofthe genera Neurospora, Aspergillus, Cephalosporium and Trichoderma, etc.

A metagenomic library refers to a genomic library constructed from theDNAs of various microorganisms present in the natural environment, whichis prepared by directly extracting DNAs from environmental samples andorganizing the resulting DNAs into a library without culturing themicroorganisms. A mutated enzyme gene library refers to a libraryprepared by introducing random mutations into DNAs encoding knownenzymes and organizing the resulting DNAs into a library.

Procedures for metagenomic library preparation can be found in, e.g., JP2007-159417 A, while procedures for mutated enzyme gene librarypreparation can be found in, e.g., Molecular Cloning: A laboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989 and Current Protocols in Molecular Biology, Supplement 1 to38, John Wiley & Sons (1987-1997). Those skilled in the art would beable to prepare the above libraries based on these documents.

More specifically, the detection method of the present invention may beaccomplished in the following manner, by way of example.

In a case where a metagenomic library or a mutated enzyme gene libraryis used as a test substance, DNAs contained in these libraries are eachtransfected into cells to prepare a library of transformants. Then, thetransformants are introduced with the compound or fluorescent substrateof the present invention to thereby cause reaction between the proteinproduced in each transformant and the compound or fluorescent substrateof the present invention, followed by measuring the wavelength andintensity of fluorescence generated as a result of the above reaction.For measurement of fluorescence, it is possible to use a fluorescencedetection instrument such as a fluorescence spectrophotometer or animager, or flow cytometry, etc.

Flow cytometry refers to a laser-based technique for measuring, e.g.,the size, DNA content, cell surface antigen distribution and/orintracellular enzymatic activity of a single cell passing through a flowcell by means of light scattering or fluorimetry. In the method of thepresent invention, a test substance and a compound whose fluorescencewill vary by the action of a nitrile-related enzyme are reacted witheach other, and fluorescence generated as a result of this reaction canbe measured using flow cytometry.

Flow cytometry may be accomplished by using a commercially availableflow cytometer in accordance with the manufacturer's instructions. Aflow cytometer is a device comprising a laser generator, an opticalsystem, a nozzle and a data processing system, and it allows automaticseparation and fluorescence analysis of fluorescence-emitting cells, aswell as their computer-aided analysis. In particular, a flow cytometerwith cell sorting functions (i.e., a cell sortor) allows high-speedscreening and sorting to collect only cells emitting desiredfluorescence. In such a cell sortor, a nozzle in the cell sortor isultrasonically vibrated to form droplets in a sheath flow, and dropletsare charged at the moment when formed from a sheath flow containingtarget cells. The charged droplets are attracted to a negatively chargeddeflection plate and collected into a tube. In this way, cells havingdesired fluorescence are screened and sorted. Examples of a commerciallyavailable flow cytometer include a BD FACSCalibur™ flow cytometer, a BDFACSAria™ III cell sortor (Nippon Becton Dickinson), etc.

In the present invention, fluorescence generated as a result of reactionbetween a test substance and the compound of the present invention ismeasured using flow cytometry, whereby cells emitting desiredfluorescence, i.e., cells containing a substance having the desiredenzymatic activity can be screened at high speed for separation orsorting. In the case of not using flow cytometry, fluorescencemeasurement can be performed on several hundreds of cells per day. Onthe other hand, in the case of using flow cytometry in a FACS(fluorescence-activated cell sorting) system, fluorescence measurementcan be performed on several tens of thousands to several million cellsper day. This means that if the number of cells processable in FACS isseveral hundred per second, the measurement of one million cells will becompleted within 1 or 2 hours, which in turn means that in the case ofusing higher performance FACS (about 7000 cells per second), themeasurement of one million cells will be completed within severalminutes.

Thus, the use of flow cytometry for fluorescence measurement in themethod of the present invention allows not only automatic and fasterdetection of the enzymatic activity of a test substance, but alsoautomatic and faster screening and sorting (high-throughput screening)of cells containing a substance having the desired enzymatic activity.

5. Kit

The present invention provides a kit for detecting the enzymaticactivity of a nitrile-related enzyme, which contains a fluorescentsubstrate comprising the above compound represented by formula (I). Thekit of the present invention may further comprise any other constituentelements (e.g., a cell lysis solution, a buffer, instructions for use)required for detecting the enzymatic activity of a nitrile-relatedenzyme, as appropriate, in addition to the fluorescent substrate of thepresent invention.

EXAMPLES

The present invention will be further described in more detail by way ofthe following illustrative examples, which are not intended to limit thescope of the invention.

Example 1 1. Synthesis of Compounds whose Fluorescence Will Vary by theAction of a Nitrile-Related Enzyme

(1) Compounds Represented by Formula (I)

As compounds whose fluorescence will vary by the action of anitrile-related enzyme, typical examples are shown below for thecompound represented by formula (I) in the present invention (wherein Merepresents a methyl group).

(2) Synthesis of Compounds Represented by Formula (I)

Compound 1 was synthesized according to scheme 1 shown below.

Compound (a) to be used as a starting material was obtained by beingpurchased from Tokyo Chemical Industry (TCI) Co., Ltd., Japan. Compound(a) was then reacted in DMF with Ac₂O and Cs₂CO₃ at room temperature for1 hour to give compound (b). Compound (b) was then dissolved in 12 NHCl, and NaNO₂ is added dropwise thereto on an ice bath, followed bystirring. After stirring for 30 minutes at 0° C., KI dissolved in waterwas added dropwise and the mixture was stirred for 10 minutes. Themixture was returned to room temperature and reacted for 1 hour to givecompound (c). Compound (c) was further reacted in THF with KCN underreflux at about 80° C. for 3 hours in the presence of CuI and Pd(PPh₃)₄as a catalyst to give compound (d). Compound (d) was reacted in MeOHwith MeONa at room temperature for 10 minutes to give compound (e).Finally, compound (e) was reacted in CsF and DMF with 1 equivalent ofCH₃I at room temperature for about 1 hour to give compound 1.

Compound 1

¹H NMR (300 MHz, CD₃OD): δ 6.82 (d, 2H, J=9.33 Hz), 6.68-6.73 (m, 4H),3.65 (s, 3H), 7.89 (d, 1H, J=1.11 Hz), 8.14 (dd, 1H, J=1.47, 8.25 Hz),8.40 (d, 1H, J=8.10 Hz) HRMS (ESI−): Calcd for [M−H]⁻, 370.0716, Found,370.0672 (−4.38 mmu)

Compound 2 was also synthesized in a manner similar to scheme 1 above.

Compound 2

¹H NMR (300 MHz, (CD₃OD): δ 3.66 (s, 3H), 6.67-6.74 (m, 4H), 6.96-6.99(m, 2H), 6.64 (d, 1H, J=8.07 Hz), 8.18 (dd, 1H J=8.07, 1.47 Hz), 8.61(d, 1H, J=1.47 Hz) HRMS (ESI−): Calcd for [M−H]⁻, 370.07155, Found,370.06831 (−3.24 mmu)

Compounds 3 and 4 were synthesized according to scheme 2 shown below.

First, compound (f) to be used as a starting material was obtained bybeing purchased from SIGMA. Compound (f) was then reacted in DMF withAc₂O and Cs₂CO₃ at room temperature for 1 hour to give compound (g)(yield: 96%). Compound (g) was then reacted in TsCl and triethylaminewith NH₄Cl supported on silica gel at room temperature for 10 minutes togive compound (h). Compound (h) was further reacted in MeOH with MeONaat room temperature for 10 minutes to give compound (i). Finally,compound (i) was reacted in CsF and DMF with 1 equivalent of CH₃I atroom temperature for 1 hour to give compounds 3 and 4 (yield: 2.3%).

Compound 3

¹H NMR (300 MHz, CD₃OD): δ 8.37 (d, 1H, J=8.07 Hz), 8.23 (dd, 1H,J=8.07, 1.47 Hz), 7.89 (d, 1H, J=1.47 Hz), 7.01 (m, 2H), 6.64 (m, 4H),3.64 (s, 3H) HRMS (ESI+): Calcd for [M+H]⁺, 390.09776, Found, 390.09473(−3.04 mmu)

Compound 4

¹H NMR (300 MHz, CD₃OD): δ 8.79 (d, 1H, J=2.20 Hz), 8.30 (dd, 1H,J=8.07, 2.20 Hz), 7.55 (d, 1H, J=8.07 Hz), 7.00 (m, 2H), 6.72 (m, 4H),3.66 (s, 1H) HRMS (ESI+): Calcd for [M+H]⁺, 390.09776, Found, 390.09457(−3.19 mmu)

Compounds 5 and 6 were synthesized according to scheme 3 shown below.

Compound (j) was reacted in H₂SO₄ with MeOH under reflux at 100° C. for1 hour to give compound (k) (yield: 8.6%). Compound (k) was then reactedin MeOH with 1 equivalent of NaOH under reflux at 100° C. for 2 hours togive compounds 5 and 6 (trace).

Compounds 5 and 6

HRMS (ESI+): Calcd for [M+H]⁺, 391.08178, Found, 391.08536 (+3.57 mmu)

2. Measurement of Fluorescence and Fluorescence Quantum Yield forCompounds

Compounds 1 to 6 obtained above were measured for their fluorescence.The results obtained are shown in FIG. 2.

In FIG. 2, “CN” represents compound 1, “CONH₂-1” represents compound 4,and “CONH₂-2” represents compound 3. Likewise, “COOH-1” represents5-COOH fluorescein, while “COOH-2” represents a mixture of compounds 5and 6.

Compounds 1 to 6 were also measured for their fluorescence quantum yield(Φ_(fl)). As a result, compounds 1 to 6 were found to have afluorescence quantum yield of 0.007, 0.161, 0.018, 0.397, 0.0729 and0.565, respectively. With respect to compounds 5 and 6, it should benoted that their fluorescence quantum yields were calculated as valuesin isolated form based on the concentration ratio determined by HPLC.

These measurement results are shown below.

TABLE 1 —CN —CONH₂ —COOH Substituent (compound 1) (compound 3) (compound5) Fluorescence 0.007 0.018 0.0729 quantum yield (Φ_(f1))

TABLE 2 —CN —CONH₂ —COOH Substituent (compound 2) (compound 4) (compound6) Fluorescence 0.161 0.397 0.565 quantum yield (Φ_(f1))

The above results indicated that compound 1 was substantiallynon-fluorescent and showed little increase in fluorescence even whenconverted into compound 3 by hydrolysis of the nitrile group (—CN) intoan amido group (—CONH₂), but it showed a large increase in fluorescencewhen converted into compound 5 by further hydrolysis of the amido (Table1). This result showed that compound 1 was able to be used as afluorescent substrate for detecting the enzymatic activity of nitrilase.

Likewise, it was also shown that compound 3 was able to be used as afluorescent substrate for detecting the enzymatic activity of amidase,because compound 3 was substantially non-fluorescent and was convertedinto fluorescent compound 5 upon hydrolysis of the amido group incompound 3.

Further, compound 2 was found to be a candidate for a fluorescentsubstrate for detecting the activity of nitrile hydratase and nitrilase,because its fluorescence intensity increased as the nitrile group (—CN)was converted into an amido group (—CONH₂) and further into a carboxylgroup (—COOH) (Table 2).

In view of the foregoing, the compounds designed and synthesized in thisexample were found to serve as fluorescent substrates for enzymaticactivity detection because of having fluorescence properties throughwhich the enzymatic reactions of nitrilase, nitrile hydratase andamidase can be visualized as changes in fluorescence.

Example 2 Nitrile Hydratase-Catalyzed Reaction

1. Preparation of Crude Nitrile Hydratase Solution (Cell-Free Extract)

Plasmid pSJ034 designed to constitutively express Rhodococcusrhodochrous strain J1-derived nitrile hydratase in Rhodococcus bacterialcells was introduced into Rhodococcus rhodochrous strain ATCC12674.pSJ034 has been prepared from plasmid pSJ023 as described in thespecification of JP 10-337185 A. pSJ023 was deposited as a transformant,R. rhodochrous ATCC12674/pSJ023 (FERM BP-6232), with the InternationalPatent Organism Depositary, the National Institute of AdvancedIndustrial Science and Technology.

Rhodococcus rhodochrous strain ATCC12674 was transformed with plasmidpSJ034 to prepare ATCC12674/pSJ034. The strain 12674 was alsotransformed with vector pK4 for use in a control experiment.

Transformation of the strain ATCC12674 was accomplished as follows.Cells of the strain ATCC12674 at the logarithmic growth phase werecollected with a centrifugal separator, washed three times with ice-coldsterilized water, and suspended in sterilized water. The plasmidsolution prepared above (1 μl) and the cell suspension (10 μl) weremixed together and cooled on ice. This mixture was transferred to acuvette for a gene transfer device, Gene Pulser (BIO RAD), andelectrically pulsed in this device at 2.0 KV at 200 OHMS. Theelectrically pulsed solution was allowed to stand under ice cooling for10 minutes and then incubated at 37° C. for 10 minutes, followed byaddition of 500 μl of MYK medium (0.5% polypeptone, 0.3% Bactoyeastextract, 0.3% Bactomalt extract, 0.2% K₂HPO₄, 0.2% KH₂PO₄). Theresulting solution was allowed to stand at 30° C. for 5 hours and thenapplied onto a 50 μg/ml kanamycin-containing MYK agar medium, followedby culturing at 30° C. for 3 days. The resulting colony was cultured andconfirmed to carry the plasmid.

The colony was cultured as follows. The colony was inoculated into akanamycin (50 mg/L)-containing MYK medium (10 ml) and pre-cultured at30° C. for 24 hours. The culture broth was taken in a volume of 1 ml andadded to 100 ml of the same medium, followed by shaking culture at 30°C. for 48 hours. The resulting culture broth was centrifuged (3,700×g,10 minutes, 4° C.) to collect the cells, which were then washed with 10mM sodium phosphate buffer (pH 7.0) and suspended in the samebuffer.

1 ml of the resulting cell suspension was homogenized for 3 minutesunder ice cooling with an ultrasonic homogenizer VP-15S (TAITEC Co.,Ltd, Japan) under the following conditions: output control 4, DUTY CYCLE40%, PULS, TIMER=B mode 10 s. The homogenate was then centrifuged(10,000×g, 5 minutes, 4° C.) and the resulting supernatant was collectedas a cell-free extract.

2. Enzymatic Reaction

10 μL of sodium phosphate buffer (pH 7.0), 75 μL of sterilized water and5 μL of 0.01 mM nitrile fluorescent substrate (compound 2: dissolved in10% DMSO) were mixed together and pre-incubated at 30° C. The cell-freeextract prepared above (10 μL) was added to initiate the reaction. Afterthe reaction at 30° C. for 1 hour, the mixture was transferred to a96-well microplate and fluorescence was detected with a fluorescenceimager (BioRad Pharos FX molecular Imager, excited at 488 nm, detectedat 530 nm).

The results obtained are shown in FIG. 3. The cell-free extract obtainedfrom the vector pK4-carrying recombinant used as a control sample showedno change in fluorescence, whereas there was a significant increase inthe intensity of fluorescence when using the nitrilehydratase-expressing recombinant.

This result indicated that the compound of the present invention wasuseful as a fluorescent substrate for detecting the enzymatic activityof a nitrile-related enzyme.

Example 3 Nitrilase-Catalyzed Reaction

1. Preparation of Crude Nitrilase Solution (Cell-Free Extract)

A recombinant E. coli, JM109/pSK002, which highly expresses Rhodococcussp. strain SK92-derived nitrilase (JP 8-173169 A) was inoculated into 1ml of a 50 μg/ml ampicillin-containing LB medium (1% Bactotryptone, 0.5%Bactoyeast extract, 0.5% NaCl) and pre-cultured at 37° C. for 7 hours.The culture broth was taken in a volume of 0.1 ml and added to 100 ml ofthe same medium (containing 50 μg/ml ampicillin and 1 mM IPTG), followedby shaking culture at 37° C. for 15 hours. The resulting culture brothwas centrifuged (3,700×g, 10 minutes, 4° C.) to collect the cells, whichwere then washed with 10 mM sodium phosphate buffer (pH 7.0) andsuspended in the same buffer. As a control strain, JM109/pUC118 wasused.

1 ml of the resulting cell suspension was homogenized for 1 minute underice cooling with an ultrasonic homogenizer VP-15S (TAITEC Co., Ltd,Japan) under the following conditions: output control 4, DUTY CYCLE 40%,PULS, TIMER=B mode 10 s. The homogenate was then centrifuged (10,000×g,5 minutes, 4° C.) and the resulting supernatant was collected as acell-free extract.

2. Enzymatic Reaction

In the same manner as shown in Example 2, the reaction was performedusing a nitrile fluorescent substrate (compound 2), followed bydetection with a fluorescence imager.

The results obtained are shown in FIG. 4. The cell-free extract obtainedfrom JM109/pUC118 used as a control sample showed no change influorescence, whereas there was a significant increase in the intensityof fluorescence when using the cell-free extract derived from thenitrilase-expressing recombinant.

This result indicated that the compound of the present invention wasuseful as a fluorescent substrate for detecting the enzymatic activityof a nitrile-related enzyme.

Example 4

10 μL of sodium phosphate buffer (pH 7.0), 75 μL of sterilized water and5 μL of 0.1 mM nitrile fluorescent substrate (compound 2: dissolved inDMSO) were mixed together and pre-incubated at 30° C. A cell-freeextract (10 μL which had been prepared in the same manner as shown inExample 3, was added to initiate the reaction. After the reaction for 24hours, the cell-free extract obtained from JM109/pUC118 used as acontrol sample showed little change in color, whereas significant greenfluorescence was observed when using the cell-free extract derived fromthe nitrilase-expressing recombinant (FIG. 5).

This result indicated that the fluorescence intensity of the fluorescentsubstrate of the present invention was significantly high enough to bedistinguishable by the naked eye from the control.

INDUSTRIAL APPLICABILITY

The compound of the present invention allows fluorescence-mediatedsimple detection of enzymatic activity. Further, when combined with FACSor the like, the present invention enables the construction of ahigh-throughput system for enzymatic activity detection.

The invention claimed is:
 1. A compound consisting of formula (I), asalt thereof, or a hydrate thereof:

wherein R¹ is —CN, —CONH₂, —CH═CH—CN: or —CH═CH—CONH₂, R² is a C 1-4alkyl group, R³, and R⁴ are each independently a hydrogen atom, ahalogen atom or a C 1-4 alkyl group, and R⁵ is a hydrogen atom, a C 1-4alkylcarbonyl group, or a C 1-4 alkylcarbonyloxymethyl group.
 2. Afluorescent substrate, according to claim 1, wherein the substrate issuitable for detecting an enzymatic activity of a nitrile-related enzymewherein the nitrile-related enzyme is selected from the group consistingof nitrilase, nitrile hydratase, and amidase.
 3. The substrate of claim2, wherein R¹ in formula (I) is —CN or —CH═CH—CN, and thenitrile-related enzyme is nitrilase or nitrile hydratase.
 4. Thesubstrate of claim 2, wherein R¹ in formula (I) is —CONH₂ or—CH═CH—CONH₂, and the nitrile-related enzyme is amidase.
 5. A kitcomprising the compound of claim
 1. 6. The kit of claim 5, furthercomprising a cell lysis solution, a buffer and/or instructions for use.7. A method for detecting enzymatic activity of a nitrile-relatedenzyme, comprising: (a) contacting the nitrile-related enzyme with thecompound of claim 1, wherein the nitrile-related enzyme is selected fromthe group consisting of nitrilase, nitrile hydratase, and amidase and(b) measuring the optical intensity of an emitted wavelength generatedas a result of step (a), wherein an increase in the intensity of theemitted wavelength positively correlates to nitrile-related enzymaticactivity.
 8. The method of claim 7, wherein R¹ in formula (I) is —CN or—CH═CH—CN, and the nitrile-related enzyme is nitrilase or nitrilehydratase.
 9. The method of claim 7, wherein R¹ in formula (I) is —CONH₂or —CH═CH—CONH₂, and the nitrile-related enzyme is amidase.
 10. Themethod of claim 7, wherein measuring step (b) comprises flow cytometry.